Method of Reducing E. coli or Salmonella Contamination of Agricultural Products

ABSTRACT

The present invention relates to a composition comprising lyophilized  Lactobacilli  and a plant-based food, wherein the composition is substantially free of animal products. The present invention also relates to methods of treating plant-based food by administration of a  Lactobacilli  and a plant medium composition. Methods of using the  Lactobacilli  and plant-based food composition to treat  E. coli  or  Salmonella  infection in an animal in need thereof and methods of using the  Lactobacilli  and plant-based food composition to reduce  E. coli  or  Salmonella  contamination in an agricultural product are also provided.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a composition comprising lyophilizedLactobacilli and a plant-based food, wherein the composition issubstantially free of animal products. The present invention alsorelates to methods of treating plant-based food by administration of aLactobacilli and a plant medium composition. Methods of using theLactobacilli and plant-based food composition to treat E. coli orSalmonella infection in an animal in need thereof and methods of usingthe Lactobacilli and plant-based food composition to reduce E. coli orSalmonella contamination in an agricultural product are also provided.

2. Background Art

Many individuals who are concerned with healthy living focus on eatingraw vegan foods. Raw foods are preferred because cooking the foodchanges the biochemical characteristics of the nutrients in the food andvegan foods are desired because many of these individuals prefer theirfood not to have any animal products or byproducts. A raw vegan diet,when it is well-balanced, offers positive health benefits to the peoplewho are following it; however, there is a major problem with potentialpathogens developing in the food because there are no chemicalpreservatives present. Usually the manufacturers of raw vegan fooddepend on refrigeration for food pathogenic bacteria containment;however, low temperatures usually do not eliminate pathogens, rather itonly keeps the pathogenic bacteria dormant. Once the food is left atroom temperature or it is consumed, the temperature of the food risescreating a positive environment for pathogenic growth. This may lead tofood poisoning and other serious illnesses.

In 2006, it was estimated that 31 major pathogens acquired in the UnitedStates caused 9.4 million episodes of foodborne illness, 55,961hospitalizations, and 1,351 deaths. Scallan, E., et al., EmergingInfectious Diseases 17:7-15 (2011). Nontyphoidal Salmonella accountedfor an estimated 1 million episodes of foodborne illnesses, 19,336hospitalizations, and 378 deaths. And, Escherichia coli (E. coli)accounted for an estimated 193,800 episodes of foodborne illness, 2,421hospitalizations, and 20 deaths.

Bacteria from the Lactobacillus family is known to have an inhibitoryeffect on food pathogens; however, most of these bacteria are grown onanimal-based medium (such as milk or meat-based medium) since theirorigin is usually from an animal or human gut. Additionally, thevitality of these bacteria grown using animal-based medium usuallydecreases significantly in a low temperature environment because theirusual environment is the body temperature of their host.

The present invention focuses on identifying specific strains ofLactobacillus among the multiple possible strains based on their naturalhabitat characteristics. The present invention also uses specificstrains of Lactobacillus as pathogenic inhibitors for vegan foods.Previously, several strains of Lactobacillus were used for their knownmicrobial application, namely yogurt production. In choosing aLactobacillus strain to use as a pathogenic inhibitor for vegan foods,three criteria are considered:

-   -   Grow well in vegetable or fruit based vegan mediums in a        laboratory environment—as it has to be potent and effective in        vegan foods.    -   Survive refrigerated temperatures—as it has to survive the usual        refrigeration of vegan foods that is common to manufacturing and        storage practices.    -   Have high inhibitory potency against E. coli and Salmonella when        inserted in contaminated vegan foods.

The bacteria kingdom consists of over 70,000 species and each carry verydifferent characteristics. Probiotics usually involve one of threegenuses of bacteria: Lactobacillus, Bifidobacteria, and Streptococcus.In each of these three genuses of bacteria, there are multiple species.

For a bacterial strain to be considered useful in the present invention,the specific bacterial strain should be of plant origin and showvitality in a plant-based environment such as vegan food.

From the known population of good bacteria, Lactobacillus delbrueckiisubsp. bulgaricus was identified as a bacteria for use in the presentinvention. Lactobacillus delbrueckii subsp. bulgaricus is foundnaturally on four different plants in Bulgaria: Cornus mas (Corneliancherry), Galanthus nivalis (snowdrop), Calendula oficinalis (commonmarigold), and Prunus spinosa (black thorn).

To be useful in the present invention, the specific bacterial strainshould survive cold temperatures. In some embodiments, the bacterialstrain useful in the present invention survive at a low temperature inits natural environment.

The strain Lactobacillus delbrueckii subsp. bulgaricus GLB 44 has beenidentified as bio-identical to bacteria found on Galanthus nivalis(snowdrop) in a northern part of Bulgaria in the mountainous region ofStaro Selo. The natural habitat for Galanthus nivalis is in amountainous region and the strain is capable of surviving in freezingtemperatures because snowdrops grow in early spring and during theirgrowth period, the temperatures fall below freezing.

It is important to note that even though the bacterial strain GLB 44 isof a known variety, it has only been shown to be useful in associationwith yogurt production in milk environments and it has not heretoforebeen tested for its survival in different plant-based mediums.Furthermore, it has neither been tested for its ability to survive inartificially refrigerated environments nor has it been tested for itseffects or E. coli or Salmonella. The present invention provides the useof this bacterial strain in compositions comprising Lactobacilli and aplant-based food wherein the composition is substantially free of animalproducts and methods of using these compositions.

BRIEF SUMMARY OF THE INVENTION

In some embodiments, the present invention provides a compositioncomprising lyophilized Lactobacilli and a plant-based food, wherein thecomposition is substantially free of animal products, and wherein theLactobacilli is Lactobacillus bulgaricus strain GLB 44.

In some embodiments, the present invention provides a method of treatingplant-based food potentially contaminated with E. coli or Salmonella,comprising treating the plant-based food with a composition comprisingLactobacilli and a plant medium, wherein the Lactobacilli isLactobacillus bulgaricus strain GLB 44.

In some embodiments, the present invention provides a method of treatingE. coli or Salmonella infection in an animal in need thereof comprisingadministering to the animal a composition comprising lyophilizedLactobacilli and a plant-based food, wherein the composition issubstantially free of animal products, and wherein the Lactobacilli isLactobacillus bulgaricus strain GLB 44.

In some embodiments, the plant-based food is a fruit juice or avegetable juice.

In some embodiments, the plant-based food is a vegetable or fruitsprayed with the Lactobacilli or is a vegetable or fruit washed in asolution that contains the Lactobacilli.

In some embodiments, the plant-based food is a vegetable or fruit andthe Lactobacilli is added during culturing.

In some embodiments, the present invention provides a method of reducingE. coli or Salmonella contamination of an agricultural product,comprising combining with or applying to the agricultural product acomposition comprising lyophilized Lactobacilli, wherein the compositionis substantially free of animal products, and wherein the Lactobacilliis Lactobacillus bulgaricus strain GLB 44.

In some embodiments, the agricultural product is a fruit juice or avegetable juice.

In some embodiments, the agricultural product is a vegetable or fruitsprayed with the Lactobacilli or is a vegetable or fruit washed with theLactobacilli.

In some embodiments, the composition contains no animal products.

In some embodiments, the vegetable or fruit is sprayed with thecomposition comprising Lactobacilli.

In some embodiments, the vegetable or fruit is washed in a solution thatcontains the Lactobacilli.

In some embodiments, the composition comprising Lactobacilli is sprayedin the form of a powder.

In some embodiments, the composition comprising Lactobacilli is sprayedin the form of a solution or suspension.

In some embodiments, the plant medium is juice from an acai, an agave,an almond, an aloe, an apple, an apricot, an arugula, an avocado, abeet, a bell pepper, a blackberry, a blue green algae, a blueberry, acarrot, a cayenne, a celery, a chia, a cilantro, a clove, a coconut, acucumber, a dandelion, a date, a fennel, a garlic, a ginger, a ginkgo, agrapefruit, a guayusa, a hemp, a jalapeno pepper, a kale, a kiwi, alemon, a lemon grass, a lime, a maca, a mandarin, an onion, an orange, aparsley, a peach, a pear, a pineapple, a raspberry, a spearmint, aspinach, a spirulina, a strawberry, a sweet potato, a tomato, a turmericroot, a watermelon, or a wheatgrass or the plant medium is soya milk,rice milk, almond milk, coffee, flax oil, herbal tea, or maple syrup.

In some embodiments, wherein after combining or applying the compositionto the agricultural product, no viable E. coli or Salmonella remains onthe agricultural product.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “substantially free” means that the content issufficiently low that no appreciable danger to humans will result fromcontact with the compositions at issue.

As used herein, animal or dairy products or byproducts mean any compoundthat was produced in or by an animal cell, whether in a living organismor in vitro. Animal products or byproducts include milk, eggs, fish, andcrustacean shellfish.

As used herein, the singular terms “a” and “the” are synonymous and usedinterchangeably with “one or more” and “at least one,” unless thelanguage and/or context clearly indicates otherwise.

As used herein, the term “comprising” means including, made up of, andcomposed of.

All numbers in this description indicating amounts, ratios of materials,physical properties of materials, and/or use are to be understood asmodified by the word “about,” except as otherwise explicitly indicated.

The term “about” as used herein includes the recited number±10%. Thus,“about ten” means 9 to 11.

The term “plant-based food” as used herein refers to a material of plantorigin that contains essential body nutrients, such as carbohydrates,fats, proteins, vitamins, or minerals, that is ingested and assimilatedby an organism to produce energy, stimulate growth, and maintain life.In some embodiments, the plant-based food is a fruit, a vegetable, agrain, or a nut.

As used herein the term “agricultural product” refers to anyagricultural commodity or product, whether raw or processed, includingany commodity or product derived from livestock, that is marketed forhuman or livestock consumption. In some embodiments, the agriculturalproduct is a plant-based food, a fiber, a fuel, a flower, an ornamentalplant, or a nursery plant. In some embodiments, the agricultural productis a plant-based food.

The term “package” as used herein refers to any type of container, box,carton, bag, or bin that can be filled with plant-based foods. A packageis designed to contain a relatively large quantity of plant-based foods.

The genus Lactobacillus contains over 180 species. In some embodiments,the Lactobacilli are of the species L. acidophilus, L. brevis, L.buchneri, L. casei, L. curvatus, L. delbrueckii, L. fermentum, L.helveticus, L. plantarum, L. reuteri, L. sakei, or L. salivarius. Insome embodiments, the Lactobacilli are from the species L. delbrueckii.In some embodiments, the Lactobacilli is Lactobacilli delbrueckiibulgaris, Lactobacilli delbrueckii lactis, Lactobacilli delbrueckiidelbrueckii, or Lactobacilli delbrueckii indicus. In some embodiments,the Lactobacilli is Lactobacilli delbrueckii bulgaris. In someembodiments, the Lactobacilli is Lactobacillus delbrueckii bulgaricusstrain GLB 44.

Lactobacillus delbrueckii subsp. bulgaricus GLB 44, was deposited withthe National Bank for Industrial Microorganisms and Cell Cultures inSofia, Bulgaria on Apr. 17, 2014, under Accession Number NBIMCC No.8814.

The present invention is based on the finding that animal products arenot required as ingredients in media for the growth of Lactobacilli, andtherefore, plant products can replace the animal products that aretypically employed in such media for the growth of Lactobacilli. Onecommonly used growth medium for the production of Lactobacillusbulgaricus is Lactobacillus bulgaricus agar base which contains caseinenzymic hydrolysate (a protein found in animal milk) and beef extract.Replacing the animal components with vegetable-based products reducesthe potential for contamination by biological molecules such as proteinsand viruses that exist in animals.

Additionally, a composition comprising Lactobacilli grown in a plantmedium can be used to replace harsh chemicals used to treat foodproducts prior to packaging. For example, baby carrots are currentlytreated with a chlorine bath before packaging to limit the risk offoodborne illnesses such as E. coli. Use of harsh chemicals can resultin degradation of the nutritional value of the food products. Replacingthe harsh chemicals with a composition of the present invention canallow for retention of valuable nutrients.

In some embodiments, the Lactobacilli is grown in a plant medium. Insome embodiments, the plant medium comprises a vegetable, a fruit, anut, or a grain. In some embodiments, the plant medium comprises thejuice of a vegetable, a fruit, a nut, or a grain. In some embodiments,the plant medium comprises carrot juice or soya milk.

In some embodiments, the media for growth of Lactobacilli containsanimal products comprising no more than 10%, no more than 9%, no morethan 8%, no more than 7%, no more than 6%, no more than 5%, no more than4%, no more than 3%, no more than 2%, no more than 1%, no more than0.9%, no more than 0.8%, no more than 0.7%, no more than 0.6%, no morethan 0.5%, no more than 0.4%, no more than 0.3%, no more than 0.2%, orno more than 0.1% of the total weight of the media. In some embodiments,the growth media is substantially free of animal products. In someembodiments, the growth media contains no animal products. In someembodiments, the growth media contains no more than 10⁹ live CFU/gram ofanimal products.

In some embodiments, the present invention provides a compositioncomprising Lactobacilli and a plant-based food. In some embodiments, thepresent invention provides a composition comprising Lactobacilli and aplant medium.

In some embodiments, the composition is a liquid, a solid, or asemi-solid. In some embodiments, the composition is lyophilized prior topackaging. In some embodiments, the composition is in the form of asolution, a suspension, an emulsion, a powder, a granule, a tablet, apellet, a multiparticulate, or a capsule. In some embodiments, thecomposition is in the form of a powder. In some embodiments, thecomposition is in the form of a solution or a suspension.

In some embodiments, the composition comprising Lactobacilli containsanimal products comprising no more than 10%, no more than 9%, no morethan 8%, no more than 7%, no more than 6%, no more than 5%, no more than4%, no more than 3%, no more than 2%, no more than 1%, no more than0.9%, no more than 0.8%, no more than 0.7%, no more than 0.6%, no morethan 0.5%, no more than 0.4%, no more than 0.3%, no more than 0.2%, orno more than 0.1% of the total weight of the media. In some embodiments,the composition comprising Lactobacilli is substantially free of animalproducts. In some embodiments, the composition comprising Lactobacillicontains no animal products. In some embodiments, the compositionscontains no more than 10⁹ live CFU/gram of animal products.

If the composition is a liquid, the concentration of Lactobacilli can bean estimate of colony-forming units per milliliter (CFU/mL). In someembodiments, the concentration of Lactobacilli in the composition isfrom 0.5 million to 1 billion CFU/mL, 0.5 million to 500 million CFU/mL,0.5 million to 400 million CFU/mL, 0.5 million to 300 million CFU/mL,0.5 million to 200 million CFU/mL, 0.5 million to 150 million CFU/mL,0.5 million to 125 million CFU/mL, 0.5 million to 100 million CFU/mL,0.5 million to 75 million CFU/mL, 0.5 million to 50 million CFU/mL, 0.5million to 10 million CFU/mL, 0.5 million to 5 million CFU/mL, 0.5million to 1 million CFU/mL, 1 million to 1 billion CFU/mL, 1 million to500 million CFU/mL, 1 million to 400 million CFU/mL, 1 million to 300million CFU/mL, 1 million to 200 million CFU/mL, 1 million to 150million CFU/mL, 1 million to 125 million CFU/mL, 1 million to 100million CFU/mL, 1 million to 75 million CFU/mL, 1 million to 50 millionCFU/mL, 1 million to 10 million CFU/mL, 1 million to 5 million CFU/mL, 5million to 1 billion CFU/mL, 5 million to 500 million CFU/mL, 5 millionto 400 million CFU/mL, 5 million to 300 million CFU/mL, 5 million to 200million CFU/mL, 5 million to 150 million CFU/mL, 5 million to 125million CFU/mL, 5 million to 100 million CFU/mL, 5 million to 75 millionCFU/mL, 5 million to 50 million CFU/mL, 5 million to 10 million CFU/mL,10 million to 1 billion CFU/mL, 10 million to 500 million CFU/mL, 10million to 400 million CFU/mL, 10 million to 300 million CFU/mL, 10million to 200 million CFU/mL, 10 million to 150 million CFU/mL, 10million to 125 million CFU/mL, 10 million to 100 million CFU/mL, 10million to 75 million CFU/mL, 10 million to 50 million CFU/mL, 50million to 1 billion CFU/mL, 50 million to 500 million CFU/mL, 50million to 400 million CFU/mL, 50 million to 300 million CFU/mL, 50million to 200 million CFU/mL, 50 million to 150 million CFU/mL, 50million to 125 million CFU/mL, 50 million to 100 million CFU/mL, 50million to 75 million CFU/mL, 100 million to 1 billion CFU/mL, 100million to 500 million CFU/mL, 100 million to 400 million CFU/mL, 100million to 300 million CFU/mL, 100 million to 200 million CFU/mL, 100million to 150 million CFU/mL, 100 million to 125 million CFU/mL, 125million to 1 billion CFU/mL, 125 million to 500 million CFU/mL, 125million to 400 million CFU/mL, 125 million to 300 million CFU/mL, 125million to 200 million CFU/mL, 125 million to 150 million CFU/mL, 150million to 1 billion CFU/mL, 150 million to 500 million CFU/mL, 150million to 400 million CFU/mL, 150 million to 300 million CFU/mL, 150million to 200 million CFU/mL, 200 million to 1 billion CFU/mL, 200million to 500 million CFU/mL, 200 million to 400 million CFU/mL, 200million to 300 million CFU/mL, 300 million to 1 billion CFU/mL, 300million to 500 million CFU/mL, 300 million to 400 million CFU/mL, 400million to 1 billion CFU/mL, 400 million to 500 million CFU/mL, or 500million to 1 billion CF U/mL.

If the composition is a solid, the concentration of Lactobacilli can bean estimate of colony-forming units per gram (CFU/g). In someembodiments, the concentration of Lactobacilli in the composition isfrom 0.5 million to 1 billion CFU/g, 0.5 million to 500 million CFU/g,0.5 million to 400 million CFU/g, 0.5 million to 300 million CFU/g, 0.5million to 200 million CFU/g, 0.5 million to 150 million CFU/g, 0.5million to 125 million CFU/g, 0.5 million to 100 million CFU/g, 0.5million to 75 million CFU/g, 0.5 million to 50 million CFU/g, 0.5million to 10 million CFU/g, 0.5 million to 5 million CFU/g, 0.5 millionto 1 million CFU/g, 1 million to 1 billion CFU/g, 1 million to 500million CFU/g, 1 million to 400 million CFU/g, 1 million to 300 millionCFU/g, 1 million to 200 million CFU/g, 1 million to 150 million CFU/g, 1million to 125 million CFU/g, 1 million to 100 million CFU/g, 1 millionto 75 million CFU/g, 1 million to 50 million CFU/g, 1 million to 10million CFU/g, 1 million to 5 million CFU/g, 5 million to 1 billionCFU/g, 5 million to 500 million CFU/g, 5 million to 400 million CFU/g, 5million to 300 million CFU/g, 5 million to 200 million CFU/g, 5 millionto 150 million CFU/g, 5 million to 125 million CFU/g, 5 million to 100million CFU/g, 5 million to 75 million CFU/g, 5 million to 50 millionCFU/g, 5 million to 10 million CFU/g, 10 million to 1 billion CFU/g, 10million to 500 million CFU/g, 10 million to 400 million CFU/g, 10million to 300 million CFU/g, 10 million to 200 million CFU/g, 10million to 150 million CFU/g, 10 million to 125 million CFU/g, 10million to 100 million CFU/g, 10 million to 75 million CFU/g, 10 millionto 50 million CFU/g, 50 million to 1 billion CFU/g, 50 million to 500million CFU/g, 50 million to 400 million CFU/g, 50 million to 300million CFU/g, 50 million to 200 million CFU/g, 50 million to 150million CFU/g, 50 million to 125 million CFU/g, 50 million to 100million CFU/g, 50 million to 75 million CFU/g, 100 million to 1 billionCFU/g, 100 million to 500 million CFU/g, 100 million to 400 millionCFU/g, 100 million to 300 million CFU/g, 100 million to 200 millionCFU/g, 100 million to 150 million CFU/g, 100 million to 125 millionCFU/g, 125 million to 1 billion CFU/g, 125 million to 500 million CFU/g,125 million to 400 million CFU/g, 125 million to 300 million CFU/g, 125million to 200 million CFU/g, 125 million to 150 million CFU/g, 150million to 1 billion CFU/g, 150 million to 500 million CFU/g, 150million to 400 million CFU/g, 150 million to 300 million CFU/g, 150million to 200 million CFU/g, 200 million to 1 billion CFU/g, 200million to 500 million CFU/g, 200 million to 400 million CFU/g, 200million to 300 million CFU/g, 300 million to 1 billion CFU/g, 300million to 500 million CFU/g, 300 million to 400 million CFU/g, 400million to 1 billion CFU/g, 400 million to 500 million CFU/g, or 500million to 1 billion CFU/g.

In some embodiments, the composition is administered to a plant-basedfood. In some embodiments, the composition is administered to anagricultural product. In some embodiments, the composition isadministered to an animal in need thereof. In some embodiments, thecomposition is administered by low pressure spraying, high pressurespraying, brushing, misting, vaporizing, volatilizing, fogging,fumigating, immersing, injecting, vapor treating, pressure treating,drenching, drip irrigating, atomizing, broadcasting, or foaming. In someembodiments, the composition is administered using an emulsion, asolution, a concentrate, a cover, a vapor, a capsule, or a microcapsule.In some embodiments, the composition is administered using a fogger, asprayer, a diffusor, a box, an envelope, a paper, a tunnel, apostharvest room, a container, a cooling room, or a refrigerator. Insome embodiments, the composition is administered by spraying. In someembodiments, the composition is administered as a solution. In someembodiments, the composition is administered as an suspension. In someembodiments, the composition is administered as a powder.

In some embodiments, the composition is administered as a solution or aspray. In some embodiments, the solution or the spray further comprisesa solvent. In some embodiments, the solvent is water.

In some embodiments, the composition is administered for 1 second to 14days, 1 second to 7 days, 1 second to 1 day, 1 second to 16 hours, 1second to 8 hours, 1 second to 1 hour, 1 second to 30 minutes, 1 secondto 10 minutes, 1 second to 1 minute, 1 second to 30 seconds, 30 secondsto 14 days, 30 seconds to 7 days, 30 seconds to 1 day, 30 seconds to 16hours, 30 seconds to 8 hours, 30 seconds to 1 hour, 30 seconds to 30minutes, 30 seconds to 10 minutes, 30 seconds to 1 minute, 1 minute to14 days, 1 minute to 7 days, 1 minute to 1 day, 1 minute to 16 hours, 1minute to 8 hours, 1 minute to 1 hour, 1 minute to 30 minutes, 1 minuteto 10 minutes, 10 minutes to 14 days, 10 minutes to 7 days, 10 minutesto 1 day, 10 minutes to 16 hours, 10 minutes to 8 hours, 10 minutes to 1hour, 10 minutes to 30 minutes, 30 minutes to 14 days, 30 minutes to 7days, 30 minutes to 1 day, 30 minutes to 16 hours, 30 minutes to 8hours, 30 minutes to 1 hour, 1 hour to 14 days, 1 hour to 7 days, 1 hourto 1 day, 1 hour to 16 hours, 1 hour to 8 hours, 8 hours to 14 days, 8hours to 7 days, 8 hours to 1 day, 8 hours to 16 hours, 16 hours to 14days, 16 hours to 7 days, 16 hours to 1 day, 1 day to 7 days, 1 day to14 days, or 7 days to 14 days.

In some embodiments, the composition comprises one or more plant-basedfoods. In some embodiments, the composition is applied to one or moreplant-based foods. In some embodiments, the composition is applied to apackage of plant-based foods. In some embodiments, the plant-based foodis a fruit, a vegetable, a grain, a nut, or combinations thereof.

In some embodiments, the plant-based food is a fruit. In someembodiments, the plant-based food is one or more fruits. In someembodiments, the plant-based food is a package of fruit. In someembodiments, plant-based food is fruit juice.

In some embodiments, the plant medium comprises a fruit.

In some embodiments, the fruit is an apple, an apricot, an avocado, abanana, a breadfruit, a bilberry, a blackberry, a blackcurrant, a bluegreen algae, a blueberry, a boysenberry, a currant, a cherry, acherimoya, a chili, a cloudberry, a coconut, a damson, a date, adragonfruit, a durian, an elderberry, a feijoa, a fig, a gooseberry, agrape, a grapefruit, a guava, a huckleberry, a jackfruit, a jettamelon,a jambul, a jujube, a kiwi fruit, a kumquat, a lemon, a lime, a loquat,a lychee, a mango, a melon, a canary melon, a cantaloupe, a honeydew, awatermelon, a rock melon, a nectarine, a nut, an orange, a clementine, amandarine, a tangerine, a papaya, a peach, a pepper, a pear, apersimmon, a tomatillo, a plum, a pineapple, a pomegranate, a pomelo, amangosteen, a quince, a raspberry, a rambutan, a redcurrant, a salalberry, a satsuma, a star fruit, a strawberry, a tamarillo, a tomato, oran ugli fruit. In some embodiments, the plant-based food is an orange oran apple. In some embodiments, the plant-based food is orange juice orapple juice.

In some embodiments, the plant-based food is a vegetable. In someembodiments, the plant-based food is one or more vegetables. In someembodiments, the plant-based food is a package of vegetables. In someembodiments, plant-based food is vegetable juice.

In some embodiments, the plant medium comprises a vegetable. In someembodiments, the plant medium comprises vegetable juice. In someembodiments, the plant medium is soya milk or carrot juice.

In some embodiments, the juice is pasteurized, fresh, or HealthcareProfessional Profile (HPP) tested.

In some embodiments, the juice does not contain significant traces ofchlorine, pesticides, or other chemical bacterial inhibitors.

In some embodiments, the temperature of the juice is above 32° F. (0°C.) when the Lactobacilli is added to the juice. In some embodiments,the temperature of the juice is below 113° F. (45° C.) when theLactobacilli is added to the juice. In the some embodiments, thetemperature of the juice is between 32° F. (0° C.) and 113° F. (45° C.)when the Lactobacilli is added to the juice.

In some embodiments, the acidity of the juice is between a pH of 3.5 to4.0. In some embodiments, the acidity of juice is at a pH less than 8.0.

In some embodiments, the vegetable is an artichoke, an arugula, anasparagus, an eggplant (aubergine), an avocado, an amaranth, an alfalfasprout, an azuki bean, a bean sprout, a black bean, a black-eyed pea, aborlotti bean, a chickpea, a green bean, a kidney bean, a lentil, a limabean, a mung bean, a navy bean, a pinto bean, a runner bean, a soybean,a pea, a bok choy, a breadfruit, a broccoflower, a broccoli, a brusselsprout, a cabbage, a calabrese, a carrot, a cauliflower, a celery, achard, a collard green, a corn, an endive, a fiddlehead, a frisee, ananise, a basil, a coriander, a chamomile, a dill, a fennel, a lavender,a lemon grass, a marjoram, an oregano, a parsley, a rosemary, a sage, athyme, a kale, a kohlrabi, a lettuce, a mushroom, a mustard green, anettle, a spinach, an okra, a chive, a garlic, a leek, an onion, ashallot, a scallion, a bell pepper, a green pepper, a chili pepper, ajalapeno pepper, a habanero pepper, a paprika pepper, a tabasco pepper,a cayenne pepper, a radicchio, a rhubarb, a beetroot, a celeriac, adaikon, a ginger, a parsnip, a rutabaga, a turnip, a radish, ahorseradish, a salsify, a skirret, an artichoke, a topinambur, an acornsquash, a butternut squash, a banana squash, a zucchini, a cucumber, adelicata, a gem squash, a hubbard squash, a pumpkin, a spaghetti squash,a tat soi, a jicama, a Jerusalem artichoke, a potato, a sweet potato, ataro, a yam, a water chestnut, or a watercress. In some embodiments, thevegetable is a carrot, a kale, a beet, or a cucumber. In someembodiments, the vegetable is carrot juice, kale juice, beet juice, orcucumber juice.

In some embodiments, the plant-based food is a grain. In someembodiments, the plant-based food is one or more grains. In someembodiments, the plant-based food is a package of grain.

In some embodiments, the plant medium comprises a grain.

In some embodiments, the grain is amaranth, barley, hulled barley,Scotch barley, pearl barley, barley flakes, barley grits, buckwheat,buckwheat groats, roasted buckwheat, buckwheat grits, corn, hominy,popcorn, millet, oats, oat groats, rolled oats, steel cut oats, quickcooking oats, instant oats, oat bran, quinoa, rice, rye, rye berries,cracked rye, rye flakes, sorghum, spelt, spelt berries, spelt flakes,teff, triticale, triticale berries, triticale flakes, wheat, wheatberries, bulgur wheat, cracked wheat, farina, semolina, wheat bran,wheat flakes, or wild rice.

In some embodiments, the plant-based food is a nut. In some embodiments,the plant-based food is one or more nuts. In some embodiments, theplant-based food is a package of nuts.

In some embodiments, the plant medium comprises a nut.

In some embodiments, the nut is an acorn, an almond, a Brazil nut, acandlenut, a cashew, a chestnut, a coconut, a hazelnut, a filbert, akola nut, a macadamia nut, a peanut, a pecan, a pili nut, a pine nut, apistachio nut, a soynut, a walnut, a black walnut, a butternut, or aheartnut.

In some embodiments, the plant-based food is an acai, an agave, analmond, an aloe, an apple, an apricot, an arugula, an avocado, a beet, abell pepper, a blackberry, a blue green algae, a blueberry, a carrot, acayenne, a celery, a chia, a chlorophyll, a cilantro, a clove, acoconut, a coffee, a cucumber, a dandelion, a date, a fennel, a flaxoil, a garlic, a ginger, a ginkgo, a grapefruit, a guayusa, a hemp, ahempseed, a hibiscus tea, a jalapeno pepper, a kale, a kiwi, a lemon, alemon grass, a lime, a maca, a mandarin, a maple syrup, an onion, anorange, a parsley, a peach, a pear, a pineapple, a raspberry, aspearmint, a spinach, a spirulina, a strawberry, a sweet potato, atahini, a tomato, a turmeric root, a watermelon, or a wheatgrass. Insome embodiments, the plant-based food is juice from an acai, an agave,an almond, an aloe, an apple, an apricot, an arugula, an avocado, abeet, a bell pepper, a blackberry, a blue green algae, a blueberry, acarrot, a cayenne, a celery, a chia, a cilantro, a clove, a coconut, acucumber, a dandelion, a date, a fennel, a garlic, a ginger, a ginkgo, agrapefruit, a guayusa, a hemp, a jalapeno pepper, a kale, a kiwi, alemon, a lemon grass, a lime, a maca, a mandarin, an onion, an orange, aparsley, a peach, a pear, a pineapple, a raspberry, a spearmint, aspinach, a spirulina, a strawberry, a sweet potato, a tomato, a turmericroot, a watermelon, or a wheatgrass or the plant medium is soya milk,rice milk, almond milk, coffee, flax oil, herbal tea, or maple syrup

In some embodiments, the present invention provides a method of treatinga pathogenic infection in an animal in need thereof comprisingadministering to the animal a composition comprising lyophilizedLactobacilli and a plant-based food. In some embodiments, theLactobacilli is Lactobacilli bulgaricus. In some embodiments, theLactobacilli is Lactobacilli bulgaricus strain GLB 44.

In some embodiments, the composition contains no animal products.

In some embodiments, the plant-based food is a fruit juice or avegetable juice.

In some embodiments, the plant-based food is a vegetable or fruitsprayed with the Lactobacilli, is a vegetable or fruit wherein theLactobacilli is added during culturing, or is a vegetable or fruitwashed in a solution that contains the Lactobacilli.

In some embodiments, the vegetable or fruit is sprayed with theLactobacilli.

In some embodiments, the vegetable or fruit is washed in a solution thatcontains the Lactobacilli.

In some embodiments, the Lactobacilli is sprayed in the form of apowder.

In some embodiments, the Lactobacilli is sprayed in the form of asolution or suspension.

In some embodiments, the plant medium is carrot juice or soya milk.

As used herein, the term “animal” includes all members of the animalkingdom including humans. In some embodiments, the animal is a mammal.In some embodiments, the animal is a human.

As used herein, “treatment” is an approach for obtaining beneficialresults, including clinical results. Beneficial or desired results caninclude, but are not limited to, alleviation or amelioration of one ormore symptoms of the condition, diminishment of the extent of thecondition, or stabilization of the state of the condition.

In some embodiments, the pathogen is a Salmonella Enterica serotype. Insome embodiments, the pathogen is Salmonella typhimurium, Salmonellaenteritidis, Salmonella newport, Salmonella hadar, Salmonellaoranienburg, Salmonella javiana, Salmonella saintpaul, Salmonellamuenchen, Salmonella agona, Salmonella I monophasic, Salmonellamontevideo, or Salmonella paratyphi. In some embodiments, the pathogenis Salmonella typhimurium. In some embodiments, the pathogen isSalmonella enteritidis.

In some embodiments, the pathogen is Bacillus cereus, Campylobacterjejuni, Clostridium botulinum, Clostridium perfringens, Cryptosporidiumparvum, Escherichia coli, Giardia lamblia, Hepatitis A, Listeriamonocytogenes, Norwalk virus, Staphylococcus, Shingella, Toxoplasmagondii, Vibrio, or Yersiniosis. In some embodiments, the pathogen isEscherichia coli.

In some embodiments, the composition is administered in the form of asolution, a suspension, an emulsion, a powder, a granule, a tablet, apellet, a multi-particulate, or a capsule.

In some embodiments, the composition is in administered in the form of asolution which is sprayed onto a vegetable, fruit, nut, or grain. Insome embodiments, the composition is administered in the form of apowder which is dusted onto a vegetable, fruit, nut, or grain. In someembodiments, the composition is administered in the form of a solutionor suspension.

In some embodiments, the plant-based food resists disease by pathogenicinfection after application for at least 1 day, at least 2 days, atleast 3 days, at least 4 days, at least 5 days, at least 6 days, atleast 7 days, at least 8 days, at least 9 days, at least 10 days, atleast 11 days, at least 12 days, at least 13 days, at least 14 days, atleast 15 days, at least 16 days, at least 17 days, at least 18 days, atleast 19 days, at least 20 days, at least 21 days, at least 22 days, atleast 23 days, at least 24 days, at least 25 days, at least 26 days, atleast 27 days, at least 28 days, at least 29 days, at least 30 days, atleast 31 days, at least two months, at least three months, at least fourmonths, at least five months, at least six months, at least sevenmonths, at least eight months, at least nine months, at least tenmonths, at least eleven months, or at least one year.

In some embodiments, after combining or applying the composition to theagricultural product, no viable E. coli or Salmonella remains on theagricultural product.

In some embodiments, the plant package is a bag, a crate, a hamper, abasket, a carton, a bulk bin, a palletized container, a flat, afiberboard container, a mesh bag, a plastic bag, a paper bag, a rigidplastic container, a plastic tray, or a clamshell.

In some embodiments, the plant package contains one or more plants. Insome embodiments, the number of plants in the plant package is at least1, at least 2, at least 5, at least 8, at least 10, at least 25, atleast 50, at least 75, at least 100, at least 125, at least 150, atleast 175, at least 200, at least 225, at least 250, at least 275, atleast 300, at least 400, at least 500, at least 600, at least 700, atleast 800, at least 900, or at least 1000.

In some embodiments, the plant or plant package is stored, afterapplication of the composition, at a temperature less than 1° C., lessthan 2° C., less than 3° C., less than 4° C., less than 5° C., less than6° C., less than 7° C., less than 8° C., less than 9° C., less than 10°C., less than 11° C., less than 12° C., less than 13° C., less than 14°C., less than 15° C., less than 16° C., less than 17° C., less than 18°C., less than 19° C., less than 20° C., less than 21° C., less than 22°C., less than 23° C., less than 24° C., less than 25° C., or less than26° C.

In some embodiments, the plant or plant package is stored, afterapplication of the composition, for at least 1 day, at least 2 days, atleast 3 days, at least 4 days, at least 5 days, at least 6 days, atleast 7 days, at least 8 days, at least 9 days, at least 10 days, atleast 11 days, at least 12 days, at least 13 days, at least 14 days, atleast 15 days, at least 16 days, at least 17 days, at least 18 days, atleast 19 days, at least 20 days, at least 21 days, at least 22 days, atleast 23 days, at least 24 days, at least 25 days, at least 26 days, atleast 27 days, at least 28 days, at least 29 days, at least 30 days, atleast 31 days, at least two months, at least three months, at least fourmonths, at least five months, at least six months, at least sevenmonths, at least eight months, at least nine months, at least tenmonths, at least eleven months, or at least one year.

In some embodiments, the plant or plant package is stored in arefrigerated environment at a temperature of between 35° F. and 50° F.,between 35 OF and 45° F., between 35° F. and 40° F., between 40 OF and50° F., between 40° F. and 45° F., or between 45 OF and 50° F. In someembodiments, the plant-based food is stored in a refrigeratedenvironment with a temperature of between 35° F. and 45° F.

The following examples are illustrative and non-limiting, of theproducts and methods described herein. Suitable modifications andadaptations of the variety of conditions, formulations, and otherparameters normally encountered in the field and which are obvious tothose skilled in the art in view of this disclosure are within thespirit and scope of the invention.

EXAMPLES Example 1 Determination of Survival of GLB 44 in Vegetable andFruit Foods Sample Solution Preparation

1 gram of Lactobacillus delbrueckii subsp. bulgaricus (GLB 44) with aconcentration 1×10⁹ colony-forming units per gram (CFU/g) is insertedinto 1 liter of each of the following 7 fruit and vegetable juices:orange juice, apple juice, carrot juice, beet juice, kale juice,pineapple juice, and cucumber juice.

GLB 44 inserted juice samples are placed in a refrigerated environmentat 40° F. 10 mL samples are collected from each of the juices at 24hours and 48 hours after the juices were placed in the refrigeratedenvironment.

Control Solution Preparation

1 gram of GLB 44 with a concentration 1×10⁹ CFU/g is inserted into 1liter of sterile milk.

Growth Medium Solution Preparation

10 mL samples from all seven of the juice solutions and the controlsolution are added to individual 100 mL milk mediums that are stabilizedat pH of 6.3 with a temperature of 98.6° F.

Measuring the Vitality of GLB 44 Via pH Measurements

The pH of each of the growth medium solutions is measured at 0 hours, 12hours, 15 hours, and 16 hours after the juice sample and the controlsample was inserted into the milk mediums. The faster the pH of thegrowth solutions falls, the more vital is the GLB 44 to that solution.

Results

The results of the pH measurements of the growth medium solutionscontaining juice samples and the control sample are shown in Table 1. Asshown in Table 1, GLB 44 survived very well in vegan mediums of fruitand vegetable juices that had been refrigerated for 24 hours and 48hours. And, GLB 44 showed a higher activity than the control sample,which was not exposed to refrigerated temperatures, in the followingrefrigerated juices: cucumber, beet, carrot, and kale. In the apple andorange juice samples, GLB 44 also showed good vitality but slightly lessthan the control sample. Therefore, GLB 44 was well tolerated in veganenvironments and withstood refrigerated temperatures. This correspondsto the bulgaricus strain which is mainly derived from the leaves of asnowdrop flower, and this strain has shown to be suitable for insertionin vegan foods that are stored in a refrigerated temperature.

TABLE 1 pH Sample 0 h 12 h 15 h 16 h Control GLB 44 6.30 4.36 4.24 4.13Orange (24 hours) 6.30 5.74 4.90 4.68 Orange (48 hours) 6.30 5.95 5.124.80 Apple (24 hours) 6.30 5.09 4.56 4.44 Apple (48 hours) 6.30 5.154.62 4.54 Carrot (24 hours) 6.30 4.24 4.13 4.06 Carrot (48 hours) 6.304.25 4.12 4.10 Kale (24 hours) 6.30 4.20 4.10 4.05 Kale (48 hours) 6.304.21 4.12 4.06 Beet (24 hours) 6.30 4.30 4.19 4.12 Beet (48 hours) 6.304.37 4.25 4.22 Cucumber (24 hours) 6.30 4.27 4.16 4.09 Cucumber (48hours) 6.30 4.28 4.15 4.13

Example 2 Pathogen Inhibitory Effect of GLB 44 Against E. coli andSalmonella Test Goal

This example was designed to determine whether Lactobacillus delbrueckiisubsp. bulgaricus (GLB 44) inhibits the presence of pathogens andprevents food poisoning in healthy people who have consumed vegetableand fruit juice containing GLB 44 together with Escherichia coli (E.coli) or Salmonella Enterica Serotype typhimurium (S. typhimurium).

Test Materials

Lyophilized Lactobacillus delbrueckii subsp. bulgaricus GLB 44 powder(concentration=1×10⁹ CFU/g);

2000 mL of organic carrot juice; and

Nutrient Agar powder (CM0003, Oxoid Limited, Basingstoke, Dartford).

Bacterial Culture Preparation

Prepare and let sit overnight a culture of E. coli and S. typhimurium at98.6° F. (37° C.) in Brain Hart Infusion or Nutrient Broth. The observedcount of live bacteria in the culture is approximately 0.6 to 0.8×10⁹CFU/mL.

Performed 10 fold dilutions in saline from 10⁻¹ to 10⁻⁶ (for example,0.5 mL of the culture in 4.5 mL of saline) using a separate tip for eachdilution.

Plate 100 μL of 10⁻⁴, 10⁻⁵, and 10⁻⁶ on nutrient agar and spread thesamples on the agar surface. Count the number of bacterial colonies thenext day. Depending of the broth media the number of live bacteria in10⁻⁶ is approximately 1,000 CFU/mL.

E. Coli Test Methodology

Control Sample: 50 mL of raw carrot juice is infected with 1,000±100live cells of E. coli. 5 identical control samples of 50 mL areprepared. To determine the exact number of live cells in the initialcontrol sample, the E. coli solution concentration of live bacterialcells measured in colony forming units (CFU) is pre-counted on NutrientAgar. Appropriate dilutions are made to transfer approximately 1,000live CFU into each 50 mL control sample of carrot juice.

Test Samples: 50 mL of raw carrot juice containing 50 mg of lyophilizedLactobacillus delbrueckii subsp. bulgaricus (GLB 44) powder (powderconcentration is 1×10⁹ CFU/g) is contaminated with 1,000±100 live cellsof E. coli. 5 identical test samples of 50 mL are prepared. The samemethod is used as in the control sample to ensure that 1,000±100 liveCFU is placed in each 50 ml test sample.

To transfer the correct amount of GLB 44 powder and ensure that it iswell mixed, the following method is used: Place 1 g of GLB 44 in 9 mL ofsterile water at room temperature. Mix this solution for 3 to 5 minutesto ensure that it is homogeneous. Pipette 0.5 mL of the mixture andtransfer to each 50 mL test sample.

The five test samples and the five control samples are placed in arefrigerated environment for 24 hours at 43 OF. This replicates themanufacturing process for juice in which the vegetables are not cleanedproperly and are contaminated with trace amounts of E. coli. In thisprocess, the juice is produced and then cooled down for at least 24hours prior to reaching the hands of the consumer.

After 24 hours at 43° F., the samples are placed in a chamber at atemperature of 98.6° F. for 48 hours. According to Department of CellBiology and Molecular Genetics, University of Maryland, College Park,Md., the pathogenic growth maximum value is achieved after 48 h. M. B.Howard and S. W. Hutcheson, Applied and Environmental Microbiology69:548-553 (2003). Also according to the European Journal of Nutrition,the mean Transit Time for healthy individuals is 62 hours. Cumming, J.H., et al., Eur. J. Nutr. 43 (Suppl. 2):118-173 (2004). Since thetransit time on average is longer than maximum pathogenic count value,it is therefore reasonable to leave the samples at 98.6° F. for 48 hoursprior to making the bacterial count. The importance of this step is tosee how both bacteria grow, and to determine whether GLB 44 has apathogenic inhibitory effect once both bacteria are put into a bodytemperature environment, after 24 hours at refrigerating temperature.

After both periods have elapsed, each of the control samples and testsamples are grown out in Nutrient Agar (CM0003, Oxoid Limited,Basingstoke, Dartford) on which the GLB 44 does not grow but E. coligrows. The count of live bacteria is estimated by plating 10 folddilutions in saline on Nutrient Agar and counting the colonies (CFU) ineach dilution.

The same procedures are repeated for higher contaminant concentration ofthe E. coli, namely:

10,000 CFU/50 mL (200 CFU/mL)

100,000 CFU/50 mL (2,000 CFU/mL)

1,000,000 CFU/50 mL (20,000 CFU/mL)

10,000,000 CFU/50 mL (200,000 CFU/mL)

Salmonella typhimurium Test Methodology

Control Sample: 50 mL of raw carrot juice is infected with 1,000±100live cells of E. coli. 5 identical control samples of 50 mL areprepared. To determine the exact number of live cells in the initialcontrol sample, the Salmonella typhimurium solution concentration oflive bacterial cells measured in colony forming units (CFU) ispre-counted on Nutrient Agar. Appropriate dilutions are made to transferapproximately 1,000 live CFU into each 50 mL control sample of carrotjuice.

Test Samples: 50 mL of raw carrot juice containing 50 mg of lyophilizedLactobacillus delbrueckii subsp. bulgaricus (GLB 44) powder (powderconcentration is 1×10⁹ CFU/g) is contaminated with 1,000±100 live cellsof Salmonella typhimurium. 5 identical test samples of 50 mL areprepared. The same method is used as in the control sample to ensurethat 1,000±100 live CFU is placed in each 50 ml test sample.

To transfer the correct amount of GLB 44 powder and ensure that it iswell mixed, the following method is used: Place 1 g of GLB 44 in 9 mL ofsterile water at room temperature. Mix this solution for 3 to 5 minutesto ensure that it is homogeneous. Pipette 0.5 mL of the mixture andtransfer to each 50 mL test sample.

The five test samples and the five control samples are placed in arefrigerated environment for 24 hours at 43° F. This replicates themanufacturing process for juice in which the vegetables are not cleanedproperly and are contaminated with trace amounts of Salmonellatyphimurium. In this process, the juice is produced and then cooled downfor at least 24 hours prior to reaching the hands of the consumer.

After 24 hours at 43° F., the samples are placed in a chamber at atemperature of 98.6° F. for 48 hours. Since the transit time on averageis longer than maximum pathogenic count value, it is thereforereasonable to leave the samples at 98.6° F. for 48 hours prior to makingthe bacterial count. The importance of this step is to see how thebacteria grows, and to determine whether GLB 44 has a pathogenicinhibitory effect once the bacteria is put into a body temperatureenvironment, after 24 hours at refrigerating temperature.

After both periods have elapsed, each of the control samples and testsamples are grown in Nutrient Agar (CM0003, Oxoid Limited, Basingstoke,Dartford) on which the GLB 44 does not grow but Salmonella typhimuriumgrows. The count of live bacteria is estimated by plating 10 folddilutions in saline on Nutrient Agar and counting the colonies (CFU) ineach dilution.

The same procedures are repeated for higher contaminant concentration ofthe Salmonella typhimurium, namely:

10,000 CFU/50 mL (200 CFU/mL)

100,000 CFU/50 mL (2,000 CFU/mL)

1,000,000 CFU/50 mL (20,000 CFU/mL)

10,000,000 CFU/50 mL (200,000 CFU/mL)

Results

The results for different concentrations of live bacteria after 48 hourswith the solutions containing juice samples and the control sample areshown in TABLE 2. As shown in TABLE 2, for the control sample the growthof S. typhimurium exceeded 10 million CFU/mL while in the carrot juicethat is populated with GLB 44 (1 million CFU/mL), there was no livepathogenic bacteria after 48 hours at 98.6 OF regardless of the initialpathogenic concentration, which varied from 20 CFU/mL to 200,000 CFU/mL.

TABLE 2 Number of live bacteria in 48 Number of live bacteria in 48hours at 98.6° F. (CFU/mL) hours at 98.6° F. (CFU/mL) with GLB 44concentration Control sample no LB 1 million CFU/mL present S.typhimurium 2692 in carrot juice 20 CFU/mL no live pathogenic bacteriagreater than 0.10 × 10⁹ 200 CFU/mL no live pathogenic bacteria greaterthan 0.10 × 10⁹ 2,000 CFU/mL no live pathogenic bacteria greater than0.10 × 10⁹ 20,000 CFU/mL no live pathogenic bacteria greater than 0.10 ×10⁹ 200,000 CFU/mL no live pathogenic bacteria greater than 0.10 × 10⁹E. coli in carrot juice 20 CFU/mL no live pathogenic bacteria greaterthan 0.12 × 10⁹ 200 CFU/mL no live pathogenic bacteria greater than 0.12× 10⁹ 2,000 CFU/mL no live pathogenic bacteria greater than 0.12 × 10⁹20,000 CFU/mL no live pathogenic bacteria greater than 0.12 × 10⁹200,000 CFU/mL no live pathogenic bacteria greater than 0.12 × 10⁹

As shown in Table 2, similar results were found with E. coli. In thecontrol sample, the E. coli exceeded 120 million CFU/ml while in thecarrot juice that is populated with GLB 44 (1 million CFU/mL), therewere no live pathogenic bacteria after 48 hours at 98.6° F. regardlessof the initial pathogenic concentration, which varied from 20 CFU/mL to200,000 CFU/mL.

As shown by the results in TABLE 2, GLB 44 performs exceptionally wellas a pathogenic inhibitor in vegan mediums. While other Lactic AcidBacteria (LAB) have shown inhibitory effects against Salmonella and E.coli, no similar results on vegan foods have been observed. Table 3summarizes scientific articles that explore the inhibitory effect ofdifferent LAB on Salmonella, E. coli or both.

While many scientific studies exist about the inhibitory effects of thedifferent Lactic Acid Bacteria, the present discovery that GLB 44completely destroys E. coli and Salmonella contamination in veganmediums is remarkable especially because GLB 44 is able to sustainitself in refrigerated temperatures for 24 hours before use. This uniqueproperty of GLB 44 is most likely due to its natural habitat being onthe leaves of snowdrops which provides for its vitality in vegan-typemediums and its ability to sustain in low temperature because in itsnatural habitat the temperature falls below the freezing point.

Example 3 Lactobacillus bulgaricus Strain GLB 44 on Salmonella and E.coli in Vegan Juice 1. Test Materials

-   -   (1) Lyophilized Lactobacillus delbrueckii subsp. bulgaricus GLB        44 powder (concentration=1×10⁹ CFU/g);    -   (2) 2000 mL of organic carrot juice;    -   (3) Nutrient Agar powder (CM0003, Oxoid Limited, Basingstoke,        Dartford).

2. Bacterial Culture Preparation

E. coli (ATCC number: 25922) and Salmonella enterica subsp. entericaserovar typhimurium (ATCC number: 14028) were grown in Tryptic soy broth(TSB, Sigma Chemical Co., St. Louis, Mo.) medium at 37° C. with shakingfor 16 hours.

Performed 10 fold dilutions from 10⁻¹ to 10⁻⁸.

Plate 1000 μl of 10⁻⁶, 10⁻⁷ and 10⁻⁸ diluted samples on nutrient agarand incubate at 37° C. for 16 hours.

3. Preparation of the Organic Carrot Juice

Sterilize the juice for 10 seconds at 273° F. (134° C.). Cool down thejuice to 43° F. (6° C.) in a refrigerator while keeping the samplecompletely sterile. Make 10 fold dilutions, and plate on TSA media at37° C. for 16 hours to check that the carrot juice is truly sterile.

4. Inoculation of the Organic Carrot Juice with E. coli or S.typhimurium

Control Samples: The plate count showed that the initial culture for E.coli was 1.2×10⁹ CFU/mL, and 1.0×10⁹ CFU/mL for S. typhimurium. 10 folddilutions were made separately from the initial cultures and 1,000±100live cells were inoculated into the 10 tubes containing 50 mL organiccarrot juice.

Test Samples: 50 mL of organic carrot juice that contains 50 mg oflyophilized L. bulgaricus powder (concentration=1×10⁹ CFU/g) iscontaminated with 1,000 live cells of E. coli or S. typhimurium.

The following method was used to transfer the correct amount of GLB 44powder: 1 g of L. bulgaricus was placed in 9 mL of sterile water at roomtemperature. the solution was mixed for 3 to 5 minutes to ensurehomogeneity. With a pipette, 0.5 mL was removed and inserted into 5tubes each for both 50 mL test samples.

5. Treatment

-   -   (1) The 10 test samples and the 10 control samples were placed        in a refrigerated environment held for 24 hours at 43° F.    -   (2) After the end of the 24 hours at 43° F., the samples were        put under a temperature of 98.6° F. for 48 hours.

6. Plate Count

After both periods have elapsed, each of the control samples and testsamples were serial diluted and plated out in Nutrient Agar.

Results

1. Test for E. coli

As shown in TABLE 3, no E. coli colony formation was detected onNutrient Agar plates for all 5 samples that were inoculated with L.bulgaricus. Conversely, control samples that were not inoculated with L.bulgaricus showed the presence of 10⁷ CFU/mL E. coli.

TABLE 3 Bacterial plate count summary for E. coli E. coli plateTreatment count (CFU/mL) E. coli with Lb-1 0 E. coli with Lb-2 0 E. coliwith Lb-3 0 E. coli with Lb-4 0 E. coli with Lb-5 0 E. coli Ck-1 9.2 ×10⁷ E. coli Ck-2 3.8 × 10⁷ E. coli Ck-3 6.4 × 10⁷ E. coli Ck-4 4.5 × 10⁷E. coli Ck-5 1.8 × 10⁷2. Test for Salmonella typhimurium

As shown in TABLE 4, no Salmonella typhimurium colony formation wasdetected on Nutrient Agar plates for all 5 samples that were inoculatedwith L. bulgaricus. Conversely, control samples that were not inoculatedwith L. bulgaricus showed the presence of 10⁵ CFU/mL Salmonellatyphimurium.

TABLE 4 Bacterial plate count summary for Salmonella typhimuriumSalmonella plate Treatment count (cfu/ml) Salmonella with Lb-1 0Salmonella with Lb-2 0 Salmonella with Lb-3 0 Salmonella with Lb-4 0Salmonella with Lb-5 0 Salmonella Ck-1 1.6 × 10⁵ Salmonella Ck-2 8.2 ×10⁵ Salmonella Ck-3 3.7 × 10⁵ Salmonella Ck-4 3.2 × 10⁵ Salmonella Ck-51.2 × 10⁵

Conclusion

The results from this experiment showed that Lactobacillus bulgaricusstrain GLB 44 inhibited 100% of the E. coli and Salmonella contaminationin vegan juice. It can also be concluded from the control samples, thatwithout the presence of the Lactobacillus bulgaricus strain GLB 44, bothE. coli and Salmonella thrive in vegan juice and small contamination ofthe food pathogens can reach high concentrations under the appropriateconditions that could cause a health hazard if consumed by humans.

Example 4 Comparison Data for 6 Different Strains of Lactobacillusbulgaricus Test Materials

Lyophilized Lactobacillus delbrueckii subsp. bulgaricus for 6 differentstrains: LBB 5, LBB 26, LBB 37, GLB 44 (NBIMCC 8814), LBB 2, and LBB 14(concentration=1×10⁶ CFU/mL);

2000 mL of organic carrot juice; and

Nutrient Agar powder (CM0003, Oxoid Limited, Basingstoke, Dartford).

Bacterial Culture Preparation

Prepare and let sit overnight a culture of E. coli and S. typhimurium at98.6° F. (37° C.) in Brain Hart Infusion or Nutrient Broth. Performed 10fold dilutions in saline from 10⁻¹ to 10⁻⁶ (for example, 0.5 mL of theculture in 4.5 mL of saline) using a separate tip for each dilution.Plate 100 μL of 10⁻⁴, 10⁻⁵, and 10⁻⁶ on nutrient agar and spread thesamples on the agar surface. Count the number of bacterial colonies thenext day. The observed count of live bacteria in the culture isapproximately 10^(4.56) CFU/mL for S. typhimurium and 10^(4.69) CFU/mLfor E. coli.

E. Coli Test Methodology

Test Samples: 50 mL of raw carrot juice containing 50 mg of thelyophilized Lactobacillus delbrueckii subsp. bulgaricus powder iscontaminated with the live cells of E. coli. 3 identical test samples of50 mL are prepared for each of the 6 strains.

The three test samples are placed in a refrigerated environment for 24hours at 39.2 OF. After 24 hours at 39.2 OF, the samples are placed in achamber at a temperature of 98.6° F. for 48 hours. After both periodshave elapsed, the test samples are grown out in Nutrient Agar (CM0003,Oxoid Limited, Basingstoke, Dartford). The count of live bacteria isestimated by plating 10 fold dilutions in saline on Nutrient Agar andcounting the colonies (CFU) in each dilution.

Salmonella typhimurium Test Methodology

Test Samples: 50 mL of raw carrot juice containing 50 mg of lyophilizedLactobacillus delbrueckii subsp. bulgaricus powder is contaminated withlive cells of Salmonella typhimurium. 3 identical test samples of 50 mLare prepared.

The three test samples are placed in a refrigerated environment for 24hours at 39.2° F. After 24 hours at 39.2° F., the samples are placed ina chamber at a temperature of 98.6° F. for 48 hours. After both periodshave elapsed, the test samples are grown out in Nutrient Agar (CM0003,Oxoid Limited, Basingstoke, Dartford). The count of live bacteria isestimated by plating 10 fold dilutions in saline on Nutrient Agar andcounting the colonies (CFU) in each dilution.

TABLE 5 Inhibition of S. typhimurium by Lactobacillus delbrueckii subsp.bulgaricus Strain LBB 5 LBB 26 LBB 37 GLB 44 LBB 2 LBB 14 NBIMCC CodeNBIMCC NBIMCC NBIMCC NBIMCC NBIMCC NBIMCC 273 285 286 8814 1273 1132Initial Contamination 10^(4.56) 10^(4.56) 10^(4.56) 10^(4.56) 10^(4.56)10^(4.56) of S. typhimurium CFU/mL CFU/mL CFU/mL CFU/mL CFU/mL CFU/mLInitial Concentration 10^(6.00) 10^(6.00) 10^(6.00) 10^(6.00) 10^(6.00)10^(6.00) of L. Bulgaricus CFU/mL CFU/mL CFU/mL CFU/mL CFU/mL CFU/mLDuration of sample 24 hours 24 hours 24 hours 24 hours 24 hours 24 hoursexposure to 4° C. (39.2° F.) Duration of sample 48 hours 48 hours 48hours 48 hours 48 hours 48 hours exposure to 37° C. (98.6° F.) Trial 1Final Contamination 10^(3.15) 10^(3.36) 10^(2.68) 0.00 10^(2.90)10^(3.98) S. typhimurium CFU/mL CFU/mL CFU/mL CFU/mL CFU/mL PercentInhibition 96.1% 93.7% 98.7% 100.0% 97.8% 73.8% Trial 2 FinalContamination 10^(3.54) 10^(3.40) 10^(2.89) 0.00 10^(2.48) 10^(3.48) S.typhimurium CFU/mL CFU/mL CFU/mL CFU/mL CFU/mL Percent Inhibition 90.5%93.1% 97.9% 100.0% 99.2% 91.7% Trial 3 Final Contamination 10^(2.45)10^(3.49) 10^(3.13) 0.00 10^(2.38) 10^(4.19) S. typhimurium CFU/mLCFU/mL CFU/mL CFU/mL CFU/mL Percent Inhibition 99.2% 91.5% 96.3% 100.0%99.3% 57.6% Average Inhibition 95.3% 92.8% 97.6% 100.0% 98.8% 74.4%(over 3 trials)

TABLE 6 Inhibition of E. Coli by Lactobacillus delbruecki subsp.bulgaricus Strain LBB 5 LBB 26 LBB 37 GLB 44 LBB 2 LBB 14 NBIMCC CodeNBIMCC NBIMCC NBIMCC NBIMCC NBIMCC NBIMCC 273 285 286 8814 1273 1132Initial Contamination 10^(4.69) 10^(4.69) 10^(4.69) 10^(4.69) 10^(4.69)10^(4.69) E. coli CFU/mL CFU/mL CFU/mL CFU/mL CFU/mL CFU/mL InitialConcentration 10^(6.00) 10^(6.00) 10^(6.00) 10^(6.00) 10^(6.00)10^(6.00) of L. bulgaricus CFU/mL CFU/mL CFU/mL CFU/mL CFU/mL CFU/mLDuration of sample 24 hours 24 hours 24 hours 24 hours 24 hours 24 hoursexposure to 4° C. (39.2° F.) Duration of sample 48 hours 48 hours 48hours 48 hours 48 hours 48 hours exposure to 37° C. (98.6° F.) (in10^(Coefficient) CFU/mL) Trial 1 Final Contamination 10^(3.54) 10^(3.81)10^(2.98) 0.00 10^(3.10) 10^(4.14) E. coli CFU/mL CFU/mL CFU/mL CFU/mLCFU/mL Percent Inhibition 93.0% 87.0% 98.1% 100.0% 97.5% 72.1% Trial 2Final Contamination 10^(3.45) 10^(3.85) 10^(3.11) 0.00 10^(3.21)10^(3.76) E. coli CFU/mL CFU/mL CFU/mL CFU/mL CFU/mL Percent Inhibition94.3% 85.7% 97.4% 100.0% 96.7% 88.4% Trial 3 Final Contamination10^(3.13) 10^(3.88) 10^(2.78) 0.00 10^(3.60) 10^(3.99) E. coli CFU/mLCFU/mL CFU/mL CFU/mL CFU/mL Percent Inhibition 97.3% 84.7% 98.8% 100.0%92.6% 80.3% Average Inhibition 94.9% 85.8% 98.1% 100.0% 95.4% 80.2%(over 3 trials)

As disclosed in TABLE 5 and TABLE 6, inoculation with GLB 44 inhibited100% of both S. typhimurium and E. coli. Conversely, inoculation withother strains of Lactobacillus delbrueckii subsp. bulgaricus showed aninhibition of between 74.4-98.8% for S. typhimurium and between80.2-98.1% for E. coli. Although this difference does not appear to belarge, it is nonetheless significant when one considers that even a fewlive bacteria could increase to very large populations very quickly.

Example 5 Pathogen Inhibitory Effect of GLB 44 Against E. coli Test Goal

This example was designed to determine whether Lactobacillus delbrueckiisubsp. bulgaricus (GLB 44) inhibits the growth of Escherichia coli (E.coli) in vegetable juice.

Test Materials

Lyophilized Lactobacillus delbrueckii subsp. bulgaricus GLB 44 powder(concentration=1×10⁹ CFU/g);

Vegetable juice (3 bottles of Juice Press Organic Complete Source (17fluid ounces each))—Juice Press Organic Complete Source is a mixedvegetable juice that includes celery, spinach, and parsley;

Tryptic Soy Agar Plates (TSA); and

Esherichia coli (E. coli)—ATCC 25922.

Bacterial Inoculum E. Coli

A previously prepared frozen tube of E. coli stock culture was used. Thebacterial concentration was 1.32×10⁹ CFU/mL.

For the E. coli and Test Samples: The frozen tube containing E. coli wasremoved from the freezer and allowed to thaw at room temperature. Thebacteria were diluted (1:10000) in sterile phosphate buffered saline toachieve a concentration of approximately 1×10⁵ CFU/mL. 50 μL of thisdilution was added to each of the control and test samples to achieve aconcentration of approximately 1×10² CFU/mL.

Lactobacillus delbrueckii Subsp. Bulgaricus (GLB 44)

2 grams of Lactobacillus delbrueckii subsp. bulgaricus (GLB 44) wasadded to 18 mL of sterile water at room temperature. The solution wasmixed using a vortex for 5 minutes to ensure the homogeneity of themixture. 0.5 mL of the prepared solution was added to each of the testsamples.

Test Procedure

The following test samples were prepared for the E. coli:

Control Samples: 5 bottles containing 50 mL each of the vegetable juice.

Test Samples: 5 bottles containing 50 mL each of boiled vegetable juiceplus Lactobacillus delbrueckii subsp. bulgaricus (GLB 44)

E coli was added to the control and test samples as described above. Allof the samples were stored in a refrigerator (4° C.) for 24 hours.Following refrigeration, the bacterial concentration for each sample wasdetermined. Serial 10 fold dilutions were made in phosphate bufferedsaline. A 0.1 mL aliquot of each dilution was plated onto TSA. The agarplates were incubated at 37° C. for 24 hours before enumeration—allcounts were recorded as CFU/mL. All test and control samples were thenplaced at 37° C. for 48 hours. Following incubation, the bacterialconcentration for each sample was determined as previously described.

Results

The results for samples of E. coli after 48 hours with the solutionscontaining test and control sample are shown in TABLE 7. As shown inTABLE 7, for the control sample the growth of E. coli exceeded 60,000CFU/mL while in the vegetable juice that was populated with GLB 44,there was no E. coli after 48 hours at 37° C.

TABLE 7 Number of E. coli Number of E. coli after 48 hours at after 48hours at 37° C. (CFU/mL) 37° C. (CFU/mL) Control sample no Test samplewith Vegetable juice GLB 44 present GLB 44 present 1 87,096 0 2 64,565 03 58,884 0 4 30,903 0 5 60,256 0 Sample Average 60,341 0 % Inhibition -100.00% 48 hours pathogenic levels by L. bulgicarus

In TABLE 8, scientific articles are provided that have studied theinhibitory effects of Lactic Acid Bacteria and in these studies whileshowing partial inhibition, have not shown complete pathogenicdestruction. It is well-known that the different strains of bacteriaeven from the same subspecies can vary substantially in theirperformance. This is attributable to the fact that the natural habitatof those species varies the similar genetic composition. For example, asshown in Table 8, a study with a different strain of Lactobacillusdelbrueckii, Lactobacillus delbrueckii subsp. bulgaricus strain DSM20081, shows that the inhibitory performance with this strain onlyreached 77% again E. coli.

Thus, due to its adaptation to its natural habitat, GLB 44 has theunique quality of being very vital in plant medium—able to survive inrefrigerated temperature and able to achieve unprecedented completeinhibitory characteristics towards E. coli and S. typhimurium.

TABLE 8 Comparative Studies Involving LAB Inhibitory Effect onSalmonella and E. coli Bacterial Cultures Involved Scientific Study andDetermination of Inhibition Result Quotation “Antagonistic ActivityLactobacillus casei “the invasion of the Caco-2 cells Exerted In Vitroand In Vivo Salmonella typhimuirum: by S. typhimurium C5 is strongly byLactobacillus casei Some conditional inhibitory reduced when thepathogen has (Strain GG) against effect - no absolute been in contactfor 1 h with the Salmonella typhimurium C5 inhibition. culture . . .When both the spent Infection,” Applied and culture supernatant and theculture Environmental Microbiology of L. casei GG were neutralized at63: 513-518 (2007). pH 7, the number of Salmonella organisms invadingcells was not different from that obtained with Salmonella organismstreated with neutralized MRS broth or with PBS.” “In vitro evaluation ofLactobacillus plantarum “Further, the anti-infective anti-infectiveactivity of a Salmonella enterica serovar characteristics of KSBT 56strain Lactobacillus plantarum Enteritidis was validated by gentamicinstrain against Evident inhibitory effect but protection assay whichrevealed Salmonella enterica serovar still limited to 80% 80% reductionin the invasion of Enteritidis,” reduction. Salmonella Enteritidis” GutPathogens 5: 11 (2013). “pH-, Lactic Acid-, and Non- Lactobacillusjohnsonii “A decrease of 3.5 logs in Lactic Acid-Dependent Lactobacillusrhamnosu viable serovar Typhimurium Activities of Lactobacillus caseiSL1344 was observed.” Probiotic Lactobacilli against Salmonellatyphimuirum Salmonella enterica Serovar Inhibitory effect present onTyphimurium,”Applied and the growth of the pathogen EnvironmentalMicrobiology without complete 71: 6008-6013 (2005). destruction.“Inhibition of Lactobacillus Acidophilus, “Data obtained from theinitial Escherichia coli O157:h7 by E. coli experiments revealedsignificant Lactobacillus Acidophilus Variable inhibitory effectvariation in the inhibitory action isolated from calves” present on thegrowth of the (% inhibition) among strains of pathogen without completecalf L. acidophilus towards E. coli destruction reaching up to O157:H7and S. aureus cultures” 96%. “In vitro anti-bacterial and Lactobacillusdelbrueckii “When two bacteria added anti-adherence effects of subsp.bulgaricus strain simultaneously (competitive Lactobacillus delbrueckiiDSM 20081, E. coli inhibition) degree of inhibition of subsp bulgaricuson Inhibitory effect present on E. coli binding by L. delbrueckiiEscherichia coli,” Res. the growth of the pathogen was 77%.” Pharm. Sci.8: 260-268 without complete (2013) destruction.

Having now fully described this invention, it will be understood bythose of ordinary skill in the art that the same can be performed withina wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or anyembodiment thereof. All patents, patent applications, and publicationscited herein are fully incorporated by reference herein in theirentirety.

1. A composition comprising from 0.5×10⁶ to 5×10⁶ CFU/mL lyophilizedLactobacilli and a plant-based food, wherein the plant-based food is avegetable juice, wherein the composition is substantially free of animalproducts, wherein the Lactobacilli is Lactobacillus bulgaricus strainGLB 44, deposited at the National Bank for Industrial Microorganisms andCell Cultures, Sofia, Bulgaria, under Accession Number 8814, and whereinthe composition is stored at a temperature between 4° C. and 37° C. 2.The composition of claim 1 containing no animal products. 3.-4.(canceled)
 5. The composition of claim 1, wherein the Lactobacilli isadded to the vegetable juice during culturing. 6.-7. (canceled)
 8. Thecomposition of claim 4, wherein the Lactobacilli is added in the form ofa powder.
 9. The composition of claim 4, wherein the Lactobacilli isadded in the form of a solution or suspension.
 10. The composition ofclaim 1, wherein the vegetable juice is juice from carrot or spinach.11.-28. (canceled)
 29. The composition of claim 4, wherein the vegetablejuice was contaminated with a pathogen before it is the lyophilizedLactobacilli are added to the vegetable juice.
 30. The composition ofclaim 29, wherein the pathogen is selected from the group consisting ofEscherichia coli, Salmonella typhimurium, and Listeria monocytogenes.